Vitamin D3 is a nutritionally modulated hormone that participates as a negative growth regulator of mammary gland development and transformation. However, the mechanism behind tissue specific vitamin D3 synthesis, metabolism, and signaling to maintain glandular homeostasis and inhibit the onset of mammary transformation remains unknown. Specifically, the contribution of mammary adipose, which accounts for ~80% of the breast, to vitamin D3 synthesis, signaling, and growth inhibition of mammary epithelial cells has yet to be investigated. Our overall objective for this R21 application is to test the concept that vitamin D3 signaling in mammary adipose tissue contributes to the negative growth regulation of mammary epithelial cells. Our central hypothesis is that the mammary adipose tissue bioactivates dietary 25-hydroxyvitamin D3 [25(OH)D3] to the active metabolite, 11,25-dihydroxyvitamin D3 [1,25(OH)2D3], and induces adipocyte Vitamin D3 Receptor (VDR) signaling along with paracrine secretion of 1,25(OH)2D3 to surrounding mammary epithelial cells to regulate growth of normal and transformed breast epithelial cells. The rationale for the proposed research is to determine the ability and impact of Vitamin D3 synthesis and VDR signaling in distinct microenvironments of the breast. Thus, obtaining evidence supporting our hypothesis that Vitamin D3 bioactivation and VDR signaling within adipose contributes to the regulation of breast epithelium that would otherwise undermine the fundability of the R01. Our proposed research is relevant to NIH's mission that pertains to nutritional science research and cancer prevention, offering a nutritional means of therapy with 25(OH)D3, bioactivated by the adipocytes and localizing therapy to breast tissue. Based upon strong preliminary data, our hypothesis will be tested by pursuing two specific aims: 1) Determine the contribution of VDR signaling in mammary adipose on ductal morphogenesis and epithelial cell growth in murine models;and 2) Establish the ability of human breast adipocytes to bioactivate 25(OH)D3 and regulate the growth of normal and transformed breast epithelial cells. Under the first aim, reciprocal mammary transplantation assays using VDR wild-type (WT) and knockout (KO) mice will be used to experimentally control the VDR status within the adipose and epithelial microenvironments. Ex-vivo cultures with primary adipocytes will also be used to investigate the significance of VDR signaling via mammary adipocytes. Under the second aim, human primary adipocytes, non- transformed and transformed mammary epithelial cells (HMECs) will be used to assess the ability of primary adipocytes to participate in growth inhibiting transformed cells by way of bioactivation of 25(OH)D3 to the active ligand 1,25(OH)2D3. The proposed research is significant because it is expected to determine the importance of vitamin D3 signaling in mammary adipose as a mediator of breast epithelial cell growth. Therefore, our studies would offer insight into the mechanism by which elevated 25(OH)D3 serum concentrations reduce the susceptibility to breast cancer. PUBLIC HEALTH RELEVANCE: The proposed research has relevance to public health because it is expected to prove the concept that breast adipose tissue, upon Vitamin D3 dietary supplementation, contributes to inhibit breast epithelial cell growth via bioactivation of 25(OH)D3 to the active ligand, 1,25(OH)2D3, and paracrine release to the surrounding epithelial cells. It is also relevant in that the results are expected to provide strong suggestive evidence regarding the mechanism(s) through which elevated vitamin D3 decreases the risk of developing breast cancer, thereby enabling subsequent studies at the R01 level. The proposed studies are of an important and under- investigated area of mammary gland biology that may serve as a nutritional preventative and/or therapeutic option to fight breast cancer.